Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.

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A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

Frontiers in Chemistry ◽

10.3389/fchem.2021.768535 ◽

2021 ◽

Vol 9 ◽

Author(s):

Birthe Meineke ◽

Johannes Heimgärtner ◽

Alexander J. Craig ◽

Michael Landreh ◽

Lindon W. K. Moodie ◽

...

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Stop Codon ◽

Bioorthogonal Chemistry ◽

Bioorthogonal Reaction ◽

Noncanonical Amino Acids ◽

Selective Reactivity ◽

Direct Incorporation ◽

Chelating Groups ◽

Amber Suppression

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

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Making Sense of “Nonsense” and More: Challenges and Opportunities in the Genetic Code Expansion, in the World of tRNA Modifications

International Journal of Molecular Sciences ◽

10.3390/ijms23020938 ◽

2022 ◽

Vol 23 (2) ◽

pp. 938

Author(s):

Olubodun Michael Lateef ◽

Michael Olawale Akintubosun ◽

Olamide Tosin Olaoba ◽

Sunday Ocholi Samson ◽

Malgorzata Adamczyk

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Protein Design ◽

Side Chains ◽

Vast Number ◽

Trna Modifications ◽

Wide Range ◽

Challenges And Opportunities ◽

Code Extension ◽

Genetic Code Expansion

The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.

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